Serving as gatekeepers that modulate ions such as potassium, sodium, and calcium, these membrane proteins play critical roles in a host of physiologic functions and pathophysiological processes.

Since ion channels operate in virtually every living cell, they represent lucrative targets for a wide range of conditions such as diabetes, cystic fibrosis, hypertension, arrhythmia, pain, and cancer.

The challenges in ion channel therapeutics include the need for higher-throughput screening and for designing more specific small molecule inhibitors that differentiate between channel physiologic states (open, closed, or inactivated). Recent advances in the field include the identification of subtype-selective blockers for the treatment of pain, creation of novel cell lines and animal models expressing specific channels, development of new high-throughput screening tools, and enhancements to safety profiling.

Select Biosciences (www.selectbiosciences.com) is holding a conference in Boston in September that will focus on ion channel targets.

New Era in Pain Medicines

Chronic pain afflicts millions of patients worldwide, and pain therapeutics represents a multibillion dollar market with significant growth expected over the next decade. Ion channels are important players in the arena of pain disorders. Icagen (www.icagen.com) is developing ion channel targeted medicines for treatment of pain and other conditions.

“Current pain-modulating medications often suffer from lack of selectivity and produce undesirable side effects,” says Douglas Krafte, Ph.D., vp of biology. “Neurons involved in sensing pain express several subtypes of sodium channels, called Nav1 subtypes. Until recently, no one could block these specific subtypes. As a result, drugs impact not only pain but also may adversely affect brain, heart, and other areas. We recently identified a small-molecule inhibitor, A-803467, for the specific subtype, called Nav1.8, which represents a major advance in this area.” The studies, done in collaboration with Abbot Laboratories (www.abbott.com), were published in the PNAS.

An important feature of the screening method is the use of whole cells, according to Dr. Krafte. “This is absolutely essential for identifying selective ion channel-based therapeutics. We use a variety of specialized assays, including high-throughput electrophysiology, to screen cells that express recombinant and native channels. The use of such cells helps to design the potential drug to interact with specific states of the channel.”

These studies pave the way for a new push to target ion channel subtypes to identify new therapeutics, according to Dr. Krafte. “The big picture is that there are a number of conditions that would benefit from being able to identify drugs that target specific subtypes. We can also use information we obtain from gene families in this area because what you determine in one member of a gene family may be useful to help in identifying small molecules to target similar subtypes in the same family.”

Portfolio of Cell Lines

Because of their critical roles in nerve and muscle functioning, ion channels not only influence pain, they also impact the central nervous system (CNS) and the cardiovascular system. Drugs that modulate these ion channels have therapeutic potential in many areas such as neuropathic pain, cardiac arrhythmia, hypertension, stroke, Parkinson’s disease, diabetes, and depression.

However, the lack of functional high-throughput screening technologies has limited ion channels as drug targets. To meet the growing demands for high-throughput tools to exploit ion channels, Millipore (www.millipore.com) is developing a series of ion channel cell lines useful for lead identification, profiling, and selectivity assays.

“Historically, there are two main recognized types of ion channels: voltage-gated and ligand-gated,” says Umesh Patel, Ph.D., director of the R&D ion channel group in the bioscience division of Millipore. “Voltage-gated channels open and close in response to changes in voltage. Therapeutically, this relates to cardiovascular areas as well as neuropathic pain. On the other hand, there is huge interest in ligand-gated channels because they impact the CNS and thus therapeutic areas such as insomnia, depression, epilepsy, and analgesia.”

Additionally, ion channels can be challenging to work with. “As the genes for ion channels are so large, they can be difficult for producing stable cells. It can take up to three years to make a validated cell line. We’ve built a portfolio of ion channel cell lines. Coupling these with the use of automated electrophysiology approaches has vastly accelerated throughput. In the past, one could only screen five to ten cells per day, but new instrumentation now allows screening of 384 cells simultaneously,” Dr. Patel says.

Millipore is developing assays using automated patch-clamp instruments for ion channel profiling to determine cardiac liabilities as well as improve on compound selectivity.

Cardiac Safety Assay Tools

A key safety issue in developing compounds that target cardiac ion channels is selectivity, reports Stephen Hess, Ph.D., scientists at Invitrogen (www.invitrogen.com). “Companies must be absolutely sure their compounds block only the necessary ion channel targets and do not compromise cardiac ion channel function, which can lead to sudden death. Studies to evaluate this specificity have been fraught with difficulties.”

Invitrogen has been building tools to use in molecular and cellular biology in this area, says Tom Livelli, vp of cellular assay systems. “We developed ion channel cell lines and further improved the performance and convenience of using these cell lines by introducing our division-arrest technology, particularly in cells used for cardiac safety.”

Invitrogen’s reagents are being utilized to construct ion channel assays. “Expression of some ion channels can result in cell toxicity and loss of expression over multiple cell passages. Invitrogen’s T-REx® expression vectors provide a method of inducible expression that keeps the gene turned off until the investigator chooses to turn it on previous to a functional assay,” Dr. Hess notes.

“Our goal is to develop validated reagents and cell lines for use in many types of tests. These tools are being validated for use in fluorescence-based, radio-ligand displacement, and manual- and high-throughput patch–clamp assays. In other words, we are building a toolbox of reagents and cell lines to use in many ways in order to make these universal assay solutions.”

HTS in Whole Animal Models

Due to its simplicity and often close resemblance to human systems, the nematode worm Caenorhabditis elegans has been used as an important option for generating drug candidates. “We are using C. elegans as a model screening system to develop therapeutics for atrial fibrillation,” says Titus Kaletta, Ph.D., director at Devgen (www.devgen.com).

According to Dr. Kaletta, C. elegans has a number of features that makes it a powerful tool. “Its genome has been completely sequenced. Also, it grows easily in culture, reproduces rapidly, can be assayed in microtiter plates, and its transparency permits fluorometric read outs. Screening in the worm increases the identification of hits with drug-like properties.”

Devgen has engineered worms to express human ion channels and screened its proprietary chemical library for inhibitors of these channels. Petra Blom, Ph.D., senior scientist, notes that their screens have disclosed novel, high-quality hits in this model. “We identified molecules that potently and selectively block the human potassium channel Kv4.3.”

“This has allowed us to quickly optimize these hits into leads with great efficacy in mammalian animal models. Indeed, one of the proprietary lead compounds is able to terminate atrial fibrillation in a canine model,” explains Dirk Leysen, Ph.D., vp of chemistry.

The company plans to select one of the Kv4.3 inhibitors as an early development candidate in the near future.

“Recent studies showed direct involvement of HCN channels in pathological hyperexcitability directly associated with angina, neuropathic pain, and epilepsy. Thus pharmacological manipulation of HCN channels represents a great potential for the development of cures to treat these debilitating diseases. Unfortunately, the preclinical pharmacology of HCN channels is severely limited, largely due to the virtual absence of an assay to perform a large-scale screening of compound libraries in anticipation to obtain a starting point for the medicinal chemistry.”

Dr. Vasilyev and collaborators developed an HCN assay-validation method that screens a smaller, focused library (thousands or tens of thousands of compounds) to model the full-scale high-throughput screening. “We constructed an Ion Channel-focused Library (ICL) based on compounds derived from corporate library substructure searches of known ion channel modulators and applied it to diversify the currently limited pharmacology of HCN channels,” he explains.

“Overall 178 ion channel standards were used for ICL construction. Library substructure-searching resulted in identification of 7,612 compounds. A single-point screening for HCN channel blockers of 4,855 available compounds resulted in 110 hits, a 2.3% hit rate, the 46 most potent of which were subsequently followed with eight-point concentration-response analysis (IonWorks HT).”

“By using the focused library approach,” Dr. Vasilyev concludes, “we have expanded the limited pharmacology of HCN channels. Additionally, the data supports the idea that focused compound libraries represent useful tools in the field of ion channel drug discovery.”

Eliminating Guesswork During Quantification

Faster, more comprehensive, and upstream-targeting ion channel assays are not without potential problems. A key aspect common to all cell- and receptor-based biological assays is the potentially misleading interpretation of results from solubility-limited compounds, according to Arnon Chait, Ph.D., president and CEO of Analiza (www.analiza.com).

“Since all such assays seek to measure concentration-dependent biological responses, an accurate determination of assay dose is essential for correct data interpretation. Many compounds in early discovery are solubility-limited, and a compound that unknowingly crashes upon introduction from stock DMSO to the aqueous buffer can yield an unacceptable, false-negative result when calculated directly from dilution factors.”

One problem is that quantitative solubility assays such as HPLC/uv require extensive method development and laborious preparation of compound calibration curves that consume precious material. Alternative, semi-quantitative approaches such as turbidity/nephelometry assays are widely known to produce erratic results.

“This approach has been used to measure solubility, quantify DMSO stock concentrations, and quantify final dose concentrations of test articles in ion channel screening assays,” Dr. Chait says. “With throughput and assay cost well comparable with discovery operations, this quantitative method has been validated with tens of thousands of compounds over several years via collaborations with big pharma and biotechs alike.

During a recent R&D collaboration with ChanTest, Analazie developed and validated a high-throughput, accurate, low-cost CRO-based technology to go hand in hand with their large panel of ion channel screening assays.

“Neurotransmitters are signaling molecules that conduct and regulate the activity of most neurological processes from consciousness to motor activity,” he says. “Uptake of neurotransmitters by neural cells is a key mechanism in regulation of these processes and thus is an important therapeutic target, in particular for areas such as depression and neurodegenerative diseases. The ability to identify and characterize selective inhibitors of the uptake process is an ongoing need for drug discovery in this area.”

Molecular Devices has developed the first live cell kinetic method for screening neurotransmitter uptake, reports Dr. Sportsman. It is based on work done by Drs. Randy Blakely and Sandy Rosenthal at Vanderbilt University.

“The Neurotransmitter Transporter Uptake Assay Kit enables a simple, robust fluorescence assay to quantify uptake of serotonin, norepinephrine, or dopamine via their respective transporters. Usually you would need three separate assays to do this, so this is a definite advance. Another advantage is that this is a homogeneous assay, meaning that there are no washing or separation steps that can introduce errors and lost time,” explains Dr. Sprotsman.

One of the ways in which the kit was validated was a test screening of the LOPAC library of pharmacologically active compounds, According to Dr. Sportsman. “This library of 1,280 compounds includes a number of inhibitors of transporters of the three neurotransmitters. We were able to correctly pick these out with good accuracy, while the vast majority of the compounds were inactive as expected. Thus this assay is not only fast, it is efficient and selective as well.”